The present invention is directed to mononucleosides and oligonucleotides that are conjugated to folic acid, related folates, antifolates and analogs thereof. The present invention further provides non-nucleosidic folate conjugates. The present invention also provides methods for preparation of nucleosidic and non-nucleosidic conjuagtes.
Protein synthesis is directed by nucleic acids through the intermediacy of messenger RNA (mRNA). Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions, such as protein synthesis, of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another
The naturally occurring events that provide the disruption of the nucleic acid function, discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989) are thought to be of two types. The first, hybridization arrest, describes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides: Miller, P. S. and Ts""O, P.O.P. (1987) Anti-Cancer Drug Design, 2:117-128, and xcex1-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
Another means by which antisense oligonucleotides disrupt nucleic acid function is by hybridization to a target mRNA, followed by enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research applications and potential therapeutic purposes. At least for therapeutic purposes, the antisense oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to exhibit their activity. However, due to the large size and unfavorable charge-size ratio of oligonucleotides, their cellular uptake is very limited. Numerous efforts have focused on increasing this membrane permeability and cellular delivery of oligonucleotides.
Efforts aimed at improving the transmembrane delivery of nucleic acids and oligonucleotides have utilized protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, and calcium phosphate mediated transformation. However, many of these techniques are limited by the types of cells in which transmembrane transport is enabled and by the conditions needed for achieving such transport. An alternative that is particularly attractive for the transmembrane delivery of oligonucleotides is the modification of the physico-chemical properties of oligonucleotides via conjugation to a molecule that facilitates transport.
One method for increasing membrane or cellular transport of oligonucleotides is the attachment of a pendant lipophilic group. Ramirez, F., Mandal, S. B. and Marecek, J. F., (1982) J. Am. Chem. Soc., 104:5483, introduced the phospholipid group 5xe2x80x2-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3xe2x80x2 and 5xe2x80x2 positions. Subsequently Shea, R. G., Marsters, J. C. and Bischofberger, N. (1990), Nuc. Acids Res., 18:3777, disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5xe2x80x2-phosphate on the 5xe2x80x2-terminus of the oligonucleotide. Certain of the Shea et. al. authors also disclosed these and other compounds in patent application PCT/US90/01002. A further glucosyl phospholipid was disclosed by Guerra, F. I., Neumann, J. M. and Hynh-Dinh, T. (1987), Tetrahedron Letters, 28:3581.
In other work, a cholesteryl group was attached to the inter-nucleotide linkage between the first and second nucleotides (from the 3xe2x80x2 terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and further in Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T. and Sarin, P. S. (1989), Proc. Natl. Acad. Sci. USA, 35 86:6553. Additional approaches to the delivery and study of oligonucleotides have involved the conjugation of a variety of other molecules and reporter groups. The aromatic intercalating agent anthraquinone was attached to the 2xe2x80x2 position of a sugar fragment of an oligonucleotide as reported by Yamana, K., Nishijima, Y., Ikeda, T., Gokota, T. Ozaki, H., Nakano, H., Sangen, O. and Shimidze, T. (1990) Bioconjugate Chem., 1:319; Lemairte, M., Bayard, B. and Lebleu, B. (1986), Proc. Natl. Acad. Sci. USA, 84:648; and Leonetti, J.-P., Degols, G. and Lebleu, B. (1990), Bioconjugate Chem., 1:149. Lysine and polylysines have also been conjugated to oligonucleotides to improve their charge-size characteristics. The poly(L-lysine) was linked to the oligonucleotide via periodate oxidation of the 3xe2x80x2-terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates are described in European Patent application 87109348.0. In this instance the lysine residue was coupled to a 5xe2x80x2 or 3xe2x80x2 phosphate of the 5xe2x80x2 or 3xe2x80x2 terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3xe2x80x2 terminus of an oligonucleotide to link a peptide to the oligonucleotide as is described by Corey, D. R. and Schultz, P. G. (1987), Science, 238:1401; Zuckermann, R. N., Corey, D. R., and Schultz, P. G. (1988), J. Am. Chem. Soc., 110:1614; and Corey, D. R., Pei, D. and Schultz, P. G. (1989), J. Am. Chem. Soc., 111:8524.
Nelson, P. S., Frye, R. A. and Liu, E. (1989), Nuc. Acids Res., 17:7187 describe a linking reagent for attaching biotin to the 3xe2x80x2-terminus of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3xe2x80x2-Amine on. It is also commercially available under the name 3xe2x80x2-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy, C. D., Richardson,. C. D. and Brousseau, R. (1991), Tetrahedron Letters, 32:879. A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5xe2x80x2-terminus of an oligonucleotide is 5xe2x80x2-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg, A. M., Gmelig-Meyling, F., Gourley, F. F., Kisch, W. J., Chrisey, L. A. and Steinberg, A. D. (1991), Antisense Research and Development, 1:161 to link fluorescein to the 5xe2x80x2-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3xe2x80x2-terminus of an oligonucleotide. Asseline, U., Delaure, M., Lancelot, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T. and Helene, C. (1984), Proc. Natl. Acad. Sci. USA, 81:3297 described linking acridine on the 3xe2x80x2-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis, J., Duncan, L. and Tregear, G. W. (1987), Tetrahedron Letters, 28:5199 report building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3xe2x80x2 hydroxyl group of the 3xe2x80x2 terminal nucleotide of the oligonucleotide. Chollet, A. (1990), Nucleosides and Nucleotides, 9:957 attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the 5xe2x80x2 terminal phosphate of an oligonucleotide. They then used the bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukin protein to the oligonucleotide.
Conjugation of lipids, reporters, peptides and other molecules to oligonucleotides is not limited to the terminal 3xe2x80x2 and 5xe2x80x2-positions. A wide variety of conjugates have also been reported in the literature wherein attachment is performed at any one or more of the 2xe2x80x2-positions on the nucleotide building blocks of the oligonucleotide. Further conjugates have also been reported wherein attachment occurs on the internucleotide linkage or on one of the atoms of the nucleobase of any one of the nucleotide units of the oligonucleotide. For example, an EDTA iron complex has been linked to the 5 position of a pyrimidine nucleoside as reported by Dreyer, G. B. and Dervan, P. B. (1985), Proc. Natl. Acad. Sci. USA, 82:968. Fluorescein has been linked to an oligonucleotide in the same manner as reported by Haralambidis, J., Chai, M. and Tregear, G. W. (1987), Nucleic Acid Research, 15:4857 and biotin in the same manner as described in PCT application PCT/US/02198. Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser, J., Cruickshank, K. A., Morrision, L. E. and Netzel, T. L. (1989), J. Am. Chem. Soc., 111:6966. A commercial reagent, Amino-Modifier-dT, from Glen Research Corporation (Sterling, Va.) can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Manoharan et al. PCT Application WO 93/07883, have also reported the conjugation of oligonucleotides with a variety of molecules such as steroids, reporter molecules, reporter enzymes, vitamins, non-aromatic lipophilic molecules, chelators, porphyrins, intercalators, peptides and proteins through the intermediacy of varied linking groups, such as w-aminoalkoxy and w-aminoalkylamino groups. Conjugation has been reported at the 3xe2x80x2-, 5xe2x80x2-, 2xe2x80x2-, internucleotide linkage and nucleobase positions of oligonucleotides. Such oligonucleotide conjugates are expected to have improved physicochemical properties that facilitated their uptake and delivery into cells as demonstrated by in vitro experiments.
The intracellular and intranuclear delivery of nucleic acids and oligonucleotides, however, is still a challenge. Most often, penetration of heretofore reported oligonucleotide conjugates has been found to be limited. This has typically been a problem because such conjugates have generally been designed to improve the passive absorption of the oligonucleotides where the size, physico-chemical properties and extra-cellular concentration of the conjugate play important limiting roles. This coupled with the limited extra-cellular stability of nucleic acids and oligonucleotides demands the development of novel conjugates that will deliver higher levels of nucleic acids and oligonucleotides into specific tissues and targeted cells. One such approach for delivering oligonucleotides, is to exploit the active transport mechanism of receptor mediated endocytosis.
Unlike many of the methods mentioned above, receptor mediated endocytotic activity can be used successfully both in vitro and in vivo. This mechanism of uptake involves the movement of ligands bound to membrane receptors into the interior of an area that is enveloped by the membrane via invagination of the membrane structure. This process is initiated via activation of a cell-surface or membrane receptor following binding of a specific ligand to the receptor. Many receptor-mediated endocytotic systems are known and have been studied, including those that recognize sugars such as galactose, mannose, mannose-6-phosphate, peptides and proteins such as transferrin, asialoglycoprotein, vitamin B12, insulin and epidermal growth factor (EGF). Receptor mediated endocytosis has been well studied and is known to be a critical pathway for the uptake and internalization of a variety of cellular nutrients. These are highly developed mechanisms because of their critical role in providing nutrients to cells and in maintaining cellular physiology. Thus many examples of the utilization of receptor mediated endocytosis pathways for the delivery of drugs, proteins, nucleic acids and other molecules to cells are known. One way in which this has been applied is the conjugation of essential nutrients that are actively transported into cells with the drug or molecule of interest. The transporters or receptors involved in the uptake are capable of recognizing the nutrient portion of the conjugate and ferrying the entire conjugate into the cell. Examples of nutrients that are actively transported into cells and that may be of use in conjugates include, but are not limited to, folic acid, vitamin B6, cholesterol and vitamin B12. Such molecules have been conjugated to macromolecules such as nucleic acids and oligonucleotides to afford conjugates that exhibit improved cellular penetration. Manorharan et al., PCT Application WO 93/07883; Low et al., U.S. Pat. Nos. 5,108,921, 5,416,016.
Many vitamins possess an acid or alcohol functionality that is readily modified for conjugation to oligonucleotides. For example, conjugation of an N-hydroxy succinimide ester of an acid moiety of retinoic acid to an amine function on a linker pendant to an oliogonucleotide resulted in a oligonucleotide-Vitamin A conjugate attached via an amide bond. Retinol has been converted to its phosphoramidite and conjugated to the 5xe2x80x2-terminus of oligonucleotides via a phosphodiester or phosphorothioate linkage. Likewise, vitamin E and vitamin B6 may also be conjugated to oligonucleotides to improve transport into cells.
Pyridoxal (vitamin B6) has specific B6-binding proteins. The role of these proteins in pyridoxal transport has been studied by Zhang and McCormick, Proc. Natl. Acad. Sci. USA, 1991 88, 10407. Zhang and McCormick also have shown that a series of N-(4xe2x80x2-pyridoxyl)amines, in which several synthetic amines were conjugated at the 4xe2x80x2-position of pyridoxal, are able to enter cells by a process facilitated by the B6 transporter. They also demonstrated the release of these synthetic amines within the cell. Other pyridoxal family members include pyridoxine, pyridoxamine, pyridoxal phosphate, and pyridoxic acid. Pyridoxic acid, niacin, pantothenic acid, biotin, folic acid and ascorbic acid can be conjugated to oligonucleotides using N-hydroxysuccinimide esters that are reactive with aminolinkers located on the oligonucleotide, as described above for retinoic acid.
Folic acid and its various forms, such as dihydrofolate and tetrahydrofolate, are essential vitamins that are crucial for the biosynthesis of nucleic acids and therefore are critical to the survival and proliferation of cells. Folate cofactors play an important role in the one-carbon transfers that are critical for the biosynthesis of pyrimidine nucleosides. Cells therefore have a sophisticated system of transporting folates into the cytoplasm. Uptake of folates occurs by two different pathways depending on the cell type. Cells expressing a carrier or transporter for folate that exhibits a low affinity (Kdxcx9c10xe2x88x926 M) for the vitamin prefer reduced over oxidized forms of folate. Cells that express membrane receptors called folate binding protein (FBP), in contrast, exhibit high binding affinity (Kdxcx9c10xe2x88x929 M) and prefer the oxidized form of the vitamin. This latter receptor is believed to mediate the uptake of folates into the cytoplasm via endocytosis.
The use of biotin conjugates and also folic acid conjugates to enhance transmembrane transport of exogenous molecules, including oligonucleotides, has been reported by Low et al., U.S. Pat. Nos. 5,108,921; 5,416,016; PCT Application WO 90/12096. Folic acid was conjugated to 3xe2x80x2-aminoalkyl-oligonucleotides at their 3xe2x80x2-terminus via carbodiimide chemistry. The multiplicity of folate receptors on membrane surfaces of most cells and the associated receptor mediated endocytotic processes were implicated in the enhanced transport of these oligonucleotide-folic acid conjugates into cells. There are however, several limitations to this approach for the conjugation of folic acid to oligonucleotides.
Folic acid and many related folates and antifolates exhibit very poor solubility that hinders the effective conjugation of folic acid to oligonucleotides and subsequent purification of oligonucleotide-folic acid conjugates. Further folic acid bears two reactive carboxylic acid groups that are just as likely to react with the terminal amino group of the 3-aminoalkyl-oligonucleotide. Thus conjugation will typically result in a mixture of a- and g-conjugates arising from the reaction of the a-carboxylate and the g-carboxylate of the glutamic acid portion of the folic acid molecule. This poses difficulties from the standpoint of characterizing the conjugate and further from the standpoint of polyglutamylation of folates. Polyglutamylation of folates is a well recognized phenomenon that has significant implications on the transport, localization and activity of folates. Since polyglutamylation rates differ significantly between the xcex1- and xcex3-carboxylates, the use of poorly defined mixtures of oligonucleotide-folate conjugates, as obtained from the Low et al. procedure, U.S. Pat. No. 5,108,921, will lead to variable transport and concentration of the conjugate. Further, the conjugation of folates onto one end of an oligonucleotide may be a disadvantage because of the known propensity of exonucleases to rapidly cleave oligonucleotides by excising the terminal residues. Also, it has been observed that oligonucleotide-folic acid conjugates prepared in this fashion are light sensitive.
Therefore, there is a clear need for new oligonucleotide-folate conjugates, and methods for their preparation, that address the shortcomings of oligonucleotide conjugates as described above. The present invention is directed to this very important end.
The present invention provides nucleosidic and non-nucleosidic folate conjugates. The present invention also provides methods for preparation of such conjugates bound to folic acid and related folates. The present invention also provides oligonucleotide-folate conjugates.
The present invention further provides novel approaches to the facile synthesis of nucleoside- and oligonucleotide-folate conjugates via a step-wise construction of the folate conjugate. The methods of the present invention are broadly applicable to the attachment of a wide variety of folates at any one of many possible sites of attachment on oligonucleotides.
The present invention provides compounds of formula XVII: 
wherein:
X4 is xe2x80x94CH(X4xe2x80x2) or a group of formula: 
X4xe2x80x2 is the side chain of a naturally-occurring or non-naturally-occurring amino acid, or a protected side chain of a naturally-occurring or non-naturally-occurring amino acid;
X5 is xe2x80x94N(X6xe2x80x2)C(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94SC(S)NHxe2x80x94, xe2x80x94SC(O)NHxe2x80x94, xe2x80x94OC(S)NHxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)(CH2)nxe2x80x94 or a bond;
n is an integer from 1 to 50;
each X6 and X6xe2x80x2 is, independently, hydrogen or a hydrocarbyl group selected from C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, C4-C14 heterocycle, C4-C14 heterocyclylalkyl, C4-C14 heteroaryl and C4-C14 heteroarylalkyl; wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy; provided that X6 is not hydrogen;
R4 is a hydroxyl group or a protected hydroxyl group;
R5xe2x80x2 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl or an amino protecting group;
R5xe2x80x3 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, formyl, aminoalkyl or hydroxymethyl;
R6 is hydrogen or an amino-protecting group; and
t is 1 or 2.
In a preferred embodiment, the X4xe2x80x2 is aminocaproic acid.
In a preferred embodiment, X6 has one of the formulas: 
wherein:
SS is a solid support;
X7 is O or CH2;
Bx is a nucleobase, C4-C14 heterocyclyl or hydrogen;
z is an integer from 1 to 50; and
u is an integer from 2 to 5.
The present invention also provides compounds having formula XVIA, XVIB, XVIC or XVID: 
wherein:
W14 has the formula: 
wherein:
X4 is xe2x80x94CH(X4xe2x80x2) or a group of formula: 
X4xe2x80x2 is the side chain of a naturally-occurring or non-naturally-occurring amino acid, or a protected side chain of a naturally-occurring non-naturally-occurring amino acid;
t is 1 or 2;
X5 is xe2x80x94N(X6xe2x80x2)C(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94SC(S)NHxe2x80x94, xe2x80x94SC(O)NHxe2x80x94, xe2x80x94OC(S)NHxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)(CH2)nxe2x80x94 or a bond;
n is an integer from 1 to 50;
each X6 and X6xe2x80x2 is, independently, a bond, hydrogen or a hydrocarbyl group selected from C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, C4-C14 heterocycle, C4-C14 heterocyclylalkyl, C4-C14 heteroaryl and C4-C14 heteroarylalkyl; wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy; provided that X6 is not hydrogen and X6xe2x80x2 is not a bond;
R1 is hydrogen or a hydroxyl protecting group;
R4 is a hydroxyl group or a protected hydroxyl group;
each R5xe2x80x2 and R40 is, independently, hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl or an amino protecting group;
R5xe2x80x3 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, formyl, aminoalkyl or hydroxymethyl;
R6 is hydrogen or an amino-protecting group;
R20 is hydrogen or a group of Formula: 
R2 is xe2x80x94N(R7)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R7 is straight or branched chain alkyl having from 1 to 10 carbons;
R3 is a phosphorus protecting group;
R21 is hydrogen, hydroxyl, fluoro or a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH or Nxe2x80x94R22xe2x80x94(R23xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
or R21 has one of the formulas: 
wherein:
y1 is 0 or 1;
each y2 is, independently, 0 to 10;
y3 is 1 to 10;
E is N(R41)(R42) or Nxe2x95x90C(R41)(R42);
each R41 and each R42 is independently H, C1-C10 alkyl, a nitogen protecting group, or R41 and R42 taken together form a nitrogen protecting group; or R41 and R42 taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;
B is a nucleobase;
M is an optionally protected internucleoside linkage;
q is from zero to about 50; and
v is from zero to about 10.
In a preferred embodiment of the present invention, X6 has one of formulas: 
wherein:
SS is a solid support;
X7 is O or CH2;
Bx is a nucleobase, C4-C14 heterocyclyl or hydrogen;
z is an integer from 1 to 50; and
u is an integer from 2 to 5.
The present invention also provides methods for the preparation of compounds of formula XVII comprising the steps of:
(a) providing a hydroxy compound of formula:
X6xe2x80x94X5xe2x80x94H
(b) protecting said hydroxyl groups of X6 with protecting groups to form a protected hydroxy compound;
(c) reacting said protected hydroxy compound with an amino-protected amino acid to form a covalently linked hydroxy compound of formula:
X6xe2x80x94X5xe2x80x94X4xe2x80x94NHxe2x80x94Ag
wherein Ag is an amino protecting group; and
(d) cleaving the amino-protecting group of said covalently linked hydroxy compound to form a hydroxy compound bearing a deprotected amino group and having formula:
X6xe2x80x94X5xe2x80x94X4xe2x80x94NH2
(e) reacting said amino group with a folate moiety; and
(f) cleaving the protecting groups on said hydroxyl groups of step (b) to form a compound of formula XVII.
The methods of the present invention also provide compounds of the formula XVII wherein R4 is a hydroxyl group protected with C1-C10 alkyl, C2-C10 alkenyl or C2-C20 alkynyl, the methods further comprising the step of deprotecting the R4 group with a deprotecting reagent. In a preferred embodiment, R4 is a methoxy group or an allyl protected hydroxyl group.
In one preferred embodiment, the deprotection reagent is an aqueous amine. In another preferred embodiment, the deprotection reagent is a combination of aqueous amine and a mercapto compound.
In one aspect of the present invention, the folate moiety of step (e) is selected such that a compound of the formula shown below is synthesized: 
In another aspect of the present invention, the folate moiety of step (e) is selected such that a compound of the formula shown below is synthesized: 
The present invention also provides methods further comprising the steps of:
(g) protecting one of said hydroxyl groups of X6 with a dimethoxytrityl group; and
(h) phosphitylating the other of said hydroxyl groups of X6.
The present invention also provides compounds of the formula: 
wherein:
X4 is xe2x80x94CH(X4xe2x80x2) or a group of formula: 
X4xe2x80x2 is the side chain of a naturally-occurring or non-naturally-occurring amino acid, or a protected side chain of a naturally-occurring or non-naturally-occurring amino acid;
X5 is xe2x80x94N(X6xe2x80x2)C(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94SC(S)NHxe2x80x94, xe2x80x94SC(O)NHxe2x80x94, xe2x80x94OC(S)NHxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)(CH2)nxe2x80x94 or a bond;
n is an integer from 1 to 50;
each X6, X6xe2x80x2 and X9 is, independently, a bond, hydrogen or a hydrocarbyl group selected from C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, C4-C14 heterocycle, C4-C14 heterocyclylalkyl, C4-C14 heteroaryl and C4-C14 heteroarylalkyl; wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy; provided that each of X6 and X6xe2x80x2 is not a bond and X6 is not hydrogen;
R4 is a hydroxyl group or a protected hydroxyl group;
R5xe2x80x2 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl or an amino-protecting group
R5xe2x80x3 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, formyl, aminoalkyl or hydroxymethyl;
R6 is hydrogen or an amino-protecting group; and
t is 1 or 2.
The present invention further provides compounds of the formula: 
wherein:
X4 is xe2x80x94CH(X4xe2x80x2) or a group of formula: 
X4xe2x80x2 is the side chain of a naturally-occurring or non-naturally-occurring amino acid, or a protected side chain of a naturally-occurring or non-naturally-occurring amino acid;
X5 is xe2x80x94N(X6xe2x80x2)C(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94SC(S)NHxe2x80x94, xe2x80x94SC(O)NHxe2x80x94, xe2x80x94OC(S)NHxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)(CH2)nxe2x80x94 or a bond;
n is an integer from 1 to 50;
each X6 and X6xe2x80x2 is, independently, hydrogen or a hydrocarbyl group selected from C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, C4-C14 heterocycle, C4-C14 heterocyclylalkyl, C4-C14 heteroaryl and C4-C14 heteroarylalkyl; wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy; provided that X6 is not hydrogen;
R4 is a hydroxyl group or a protected hydroxyl group;
R5xe2x80x2 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl or an amino-protecting group
R5xe2x80x3 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, formyl, aminoalkyl or hydroxymethyl;
R6 is hydrogen or an amino protecting group; and
t is 1 or 2.
The present invention also provides compounds of the formula XVIA, XVIB, XVIC or XVID: 
wherein:
W14 has the formula: 
wherein:
X4 is xe2x80x94CH(X4xe2x80x2) or a group of formula: 
X4xe2x80x2 is the side chain of a naturally-occurring or non-naturally-occurring amino acid, or a protected side chain of a naturally-occurring or non-naturally-occurring amino acid;
t is 1 or 2;
X5 is xe2x80x94N(X6xe2x80x2)C(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94SC(S)NHxe2x80x94, xe2x80x94SC(O)NHxe2x80x94, xe2x80x94OC(S)NHxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)(CH2)nxe2x80x94 or a bond;
n is an integer from 1 to 50;
each X6, X6xe2x80x2 and X9 is, independently, a bond, hydrogen or a hydrocarbyl group selected from C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, C4-C14 heterocycle, C4-C14 heterocyclylalkyl, C4-C14 heteroaryl and C4-C14 heteroarylalkyl; wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy; provided that each X6 and X9 is not hydrogen and X6xe2x80x2 is not a bond;
R1 is hydrogen or a hydroxyl protecting group;
R4 is a hydroxyl group or a protected hydroxyl group;
each R5xe2x80x2 and R40 is, independently, hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl or an amino-protecting group
R5xe2x80x3 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, formyl, aminoalkyl or hydroxymethyl;
R6 is hydrogen or an amino protecting group;
R20 is hydrogen or a group of formula: 
R2 is xe2x80x94N(R7)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R7 is straight or branched chain alkyl having from 1 to carbons;
R3 is a phosphorus protecting group;
R21 is hydrogen, hydroxyl, fluoro or a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH or Nxe2x80x94R22xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
or R21 has one of the formulas: 
wherein:
y1 is 0 or 1;
each y2 is, independently, 0 to 10;
y3 is 1 to 10;
E is N (R41)(R42) or Nxe2x95x90C (R41)(R42);
each R41 and each R42 is independently H, C1-C10 alkyl, a nitogen protecting group, or R41 and R42 taken together form a nitrogen protecting group; or R41 and R42 taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;
B is a nucleobase;
M is an optionally protected internucleoside linkage;
q is from zero to about 50; and
v is from zero to about 10.
The present invention further provides compounds of the formula XVIA, XVIB, XVIC or XVID: 
wherein:
W14 has the formula: 
wherein:
X4 is xe2x80x94CH(X4xe2x80x2) or a group of formula: 
X4xe2x80x2 is the side chain of a naturally-occurring or non-naturally-occurring amino acid, or a protected side chain of a naturally-occurring or non-naturally-occurring amino acid;
t is 1 or 2;
X5 is xe2x80x94N(X6xe2x80x2)C(O)xe2x80x94, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94OC(O)NHxe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94SC(S)NHxe2x80x94, xe2x80x94SC(O)NHxe2x80x94, xe2x80x94OC(S)NHxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)(CH2)nxe2x80x94 or a bond;
n is an integer from 1 to 50;
each X6 and X6xe2x80x2 is, independently, a bond, hydrogen or a hydrocarbyl group selected from C1-C10 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, C5-C14 fused cycloalkyl, C4-C14 heterocycle, C4-C14 heterocyclylalkyl, C4-C14 heteroaryl and C4-C14 heteroarylalkyl; wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy; provided that X6 is not hydrogen and X6xe2x80x2 is not a bond;
R1 is hydrogen or a hydroxyl protecting group;
R4 is a hydroxyl group or a protected hydroxyl group;
each R5xe2x80x2 and R40 is, independently, hydrogen, C2-C20 alkyl, C2-C10 alkenyl, C2-C20 alkynyl, C6-C14 aryl or an amino-protecting group
R5xe2x80x3 is hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C220 alkynyl, C6-C14 aryl, C6-C14 aralkyl, C3-C14 cycloalkyl, formyl, aminoalkyl or hydroxymethyl;
R6 is hydrogen or an amino protecting group;
R20 is hydrogen or a group of formula: 
R2 is xe2x80x94N(R7)2, or a heterocycloalkyl or eterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R7 is straight or branched chain alkyl having from 1 to 10 carbons;
R3 is a phosphorus protecting group;
R21 is hydrogen, hydroxyl, fluoro or a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH or Nxe2x80x94R22xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
or R21 has one of the formulas: 
wherein:
y1 is 0 or 1;
each y2 is, independently, 0 to 10;
y3 is 1 to 10;
E is N(R41)(R42) or Nxe2x95x90C(R41)(R42);
each R41 and each R42 is independently H, C1-C10 alkyl, a nitogen protecting group, or R41 and R42 taken together form a nitrogen protecting group; or R41 and R42 taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;
B is a nucleobase;
M is an optionally protected internucleoside linkage;
q is from zero to about 50; and
v is from zero to about 10.
In accordance with the present invention, synthetic methods are provided comprising the steps of:
(a) providing a compound of formula IA, IB, IC or ID: 
wherein:
W1 is a linking group, O, NH or S, with a linking group being preferred;
R1 is H or a hydroxyl protecting group;
B is a nucleobase;
each R21 is H, OH, F, or a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH, or Nxe2x80x94R22xe2x80x94(R23)v 
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
v is from 0 to about 10;
or R21 has one of the formulas: 
wherein:
y1 is 0 or 1;
each y2 is, independently, 0 to 10;
y3 is 1 to 10;
E is N(R41)(R42) or Nxe2x95x90C(R41)(R42);
each R40, R41 and R42 is independently H, C1-C10 alkyl, a nitrogen protecting group, or R41 and R42 taken together form a nitrogen protecting group; or R41 and R42 taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;
q is from 0 to about 50;
M is an optionally protected internucleoside linkage;
(b) reacting said compound of formula I with a compound of formula II: 
wherein:
R30 is an amino protecting group;
X3 is xe2x80x94CH(Z1)xe2x80x94 or a group of formula XI: 
Z1 is the sidechain of a naturally occurring amino acid, or a protected sidechain of a naturally occurring amino acid, wherein the amino acid is preferably glutamic acid;
p is 1 or 2, with 2 being preferred; to form a compound of formula IVA, IVB, IVC, or IVD: 
wherein:
W4 has the formula: 
and treating said compound of formula IVA, IVB, IVC or IVD with a deprotecting reagent to form a compound of formula VA, VB, VC or VD: 
wherein W5 has the formula: 
In some preferred embodiments, the methods of the invention further comprise condensing said compound of formula V with a compound of formula VI: 
wherein
R5 is H or an amino protecting group;
R6 is H or an amino protecting group; to form a compound of formula VIIA, VIIB, VIIC, OR VIID: 
wherein W7 has the formula: 
Some preferred embodiments of the methods of the invention further comprise contacting said compound of formula VIIA or VIID with a phosphitylating reagent to form a compound of formula VIIIA or VIIIA-D: 
wherein We has the formula: 
In some preferred embodiments, said compound of formula VI is prepared by the steps of reacting a compound of formula IX: 
with a compound of formula X: 
and treating the product of said reaction with a protecting group reagent to form said compound of formula VI.
Also provided in accordance with the present invention are methods for the preparation of a folic acid derivative comprising the steps of reacting folic acid: 
with a reagent effective to form pterin aldehyde: 
Preferably, the methods further comprise protecting the amino group of said pterin aldehyde.
The present invention also provides compounds having the formula XIIIA, XIIB, XIIIC or XIIID: 
wherein:
W13 has the formula: 
R1 is H or a hydroxyl protecting group;
B, is a nucleobase;
each R21 is H, OH, F, or a group of formula Zxe2x80x94R22xe2x80x94(R23)v;
Z is O, S, NH or Nxe2x80x94R22xe2x80x94(R23)v;
R22 is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl;
R23 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmacokinetic properties of oligonucleotides;
or R21 has one of the formulas: 
wherein:
y1 is 0 or 1;
each y2 is, independently, 0 to 10;
y3 is 1 to 10;
E is N(R41)(R42) or Nxe2x95x90C(R41)(R42);
each R40, R41 and R42 is independently H, C1-C10 alkyl, a nitogen protecting group, or R41 and R42 taken together form a nitrogen protecting group; or R41 and R42 taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;
v is from 0 to about 10;
q is from 0 to about 50;
M is an optionally protected internucleoside linkage;
W1 is a linking group, O, NH or S, with a linking group being preferred;
R20 is H or a group of formula: 
R2 is xe2x80x94N(R7)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R7 is straight or branched chain alkyl having from 1 to 10 carbons;
R3 is a phosphorus protecting group;
n is from 1 to about 10;
R5 is H or an amino protecting group;
R6 is H or an amino protecting group;
X3 is xe2x80x94CH(Z1)xe2x80x94 or a group of formula XI: 
Z1 is the sidechain of a naturally occurring amino acid, or a protected sidechain of a naturally occurring amino acid, wherein the amino acid is preferably glutamic acid;
p is 1 or 2, with 2 being preferred; and
R4 is a hydroxyl group, or a protected hydroxyl group.
The present invention also provides compositions comprising a compound of formula XIIIA-D wherein X3 has the formula XI and p is 2, said composition being substantially free of a compound of formula XIVA, XIVB, XIVC, or XIVD: 
wherein:
W14 has the formula: 
Also provided in accordance with the present invention are compositions comprising a compound of formula XIIIA-D wherein X3 has the formula XII and m is 2, said composition being substantially free of a compound of formula XVA, XVB, XVC or XVD: 
wherein W15 has the formula: 
The present invention further provides compounds having the formula XVIA, XVIB, XVIC or XVID: 
wherein:
W16 has the formula: 
wherein the constituent variables are as defined above.
Also provided in accordance with the present invention are compounds having the formula XVIIA, XVIIB, XVIIC or XVIID: 
wherein:
W17 has the formula: 
wherein the constituent variables are as defined above.
In some preferred embodiments of the foregoing methods and compounds, X3 is a group of formula XI: 
wherein:
p is 1 or 2, with 2 being preferred;
R4 is a hydroxyl group, or a protected hydroxy group;
or X3 is a group of formula XII: 
wherein m is 1 or 2, with 2 being preferred.
In some preferred embodiments of the foregoing compounds and methods, q is 0.
In further preferred embodiments of the foregoing compounds and methods, X3 is a group of formula XI, preferably wherein p is 2.
In further preferred embodiments of the foregoing compounds and methods, X3 is a group of formula XII, preferably wherein m is 2.
In preferred embodiments of the foregoing compounds and methods, W1 has the formula xe2x80x94Oxe2x80x94(CH2)nxe2x80x94NHxe2x80x94, where n is from 1 to about 10, with 6 being preferred.
In some preferred embodiments of the foregoing compounds and methods, R1 is dimethoxytrityl. In further preferred embodiments, R4 is t-butoxy. In still further preferred embodiments, R5 is trifluoroacetoyl. In further preferred embodiments, R6 is xe2x80x94C(xe2x95x90O)xe2x80x94CH(CH3)2. In yet further preferred embodiments, R30 is fluorene-9-yl methoxycarbonyl.
In some prefered embodiments of the foregoing compounds and methods, R1 is dimethoxytrityl, W1 has the formula xe2x80x94Oxe2x80x94(CH2)nxe2x80x94NHxe2x80x94 where n is 6, X3 has the formula XI where p is 2, R4 is t-butoxy, R5 is trifluoroacetoyl, R6 is xe2x80x94C(xe2x95x90O)xe2x80x94CH(CH3)2, and R30 is FMOC.